The capacity for sustained self-renewal is one of the defining features of hematopoietic stem cells (HSCs). However, the molecular bases for HSC self-renewal remain obscure. We have previously reported that HSC expansion is profoundly affected by Hoxb4 overexpression and more recently developed HOXB4- based reagents and strategies which provide tools for deepening our knowledge of self-renewal mechanisms, and for achievement of clinically useful levels of HSC expansion. Four aims are proposed in this first renewal application: 1-To enhance the potential of HSCs to expand in vitro. This will be investigated first by varying Hoxb4 and Pbxl levels in HSCs as recently reported with our in vivo studies and by testing novel Hox-containing fusion genes with enhanced potential for HSC expansion. The contribution of growth factors and precursor differentiation stage (e.g., HSC vs MPP, etc) to HOXB4-driven HSC expansion will also be assessed. 2-To develop and test clinically-relevant strategies aimed at achieving a maximal expansion of HSCs in vitro. This includes the generation of soluble reagents that will interfere with the function of PBX1 and by testing the function of stabilized HOXB4 proteins. Studies with human HSCs will be conducted and effects of the manipulation evaluated by xenotransplantation using NOD/SCID mice and fetal sheep as recipients. 3-To identify a HOXB4-containing "HSC-self-renewal protein complex" and determine the role of the newly identified proteins in HSC self-renewal. Strategies include the utilization of a functional tandem affinity purification (TAP)-tagged HOXB4 and isolation of the complex in relevant cells. Identified proteins will be functionally validated using RNA interference. 4-To identify target genes to HOXB4 in purified mouse HSCs. This aim combines cell purification of newly characterized HSC populations with microarray analysis from small cell numbers and preliminary target gene validation.